Corn event mzdt09y

ABSTRACT

A novel transgenic corn event designated MZDT09Y is disclosed. The invention relates to nucleic acids that are unique to event MZDT09Y and to methods of detecting the presence of event MZDT09Y based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the MZDT09Y event and of genomic sequences flanking the insertion site. The invention further relates to corn plants comprising the transgenic genotype of event MZDT09Y and to methods for producing a corn plant by crossing a corn plant comprising the MZDT09Y genotype with itself or another corn variety. Seeds of corn plants comprising the MZDT09Y genotype are also objects of the invention.

BACKGROUND OF THE INVENTION

The increasing world population and the dwindling supply of arable landavailable for agriculture fuels the need for research in the area ofincreasing the efficiency of agriculture. Conventional means for cropand horticultural improvements utilize selective breeding techniques toidentify plants having desirable characteristics. However, suchselective breeding techniques have several drawbacks, namely that thesetechniques are often labor intensive and result in plants that oftencontain heterogeneous genetic components that may not always result inthe desirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant's genome. Such technology has the capacity to deliver crops orplants having various improved economic, agronomic or horticulturaltraits.

SUMMARY OF THE INVENTION

The following Summary lists several embodiments of the invention subjectmatter, and in many cases lists variations and permutations of theseembodiments. This Summary is merely exemplary of the numerous and variedembodiments. Mention of one or more representative features of a givenembodiment is likewise exemplary. Such an embodiment can typically existwith or without the feature(s) mentioned; likewise, those features canbe applied to other embodiments of the invention, whether listed in thisSummary or not. To avoid excessive repetition, this Summary does notlist or suggest all possible combinations of such features.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The invention provides nucleotide sequences that when transgenicallyexpressed in a plant increases plant vigor, yield and/or biomass as wellas increased stress tolerance. It was discovered that the T6PP proteinsdescribed herein comprise modifications which are significantlyassociated with increased yield and/or increased tolerance to stresswhen transgenically expressed in a plant.

In one embodiment, the present invention encompasses a nucleic acidmolecule, preferably isolated, comprising a nucleotide sequence that isunique to corn event MZDT09Y. The nucleotide sequence may comprise anyone of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any ofthe complements thereof. The nucleic acid molecule is comprised in acorn seed deposited at the American Type Culture Collection under theaccession number PTA-13025.

In another embodiment, the present invention encompasses a pair ofpolynucleotide primers comprising a first polynucleotide primer and asecond polynucleotide primer which function together in the presence ofan event MZDT09Y DNA template in a sample to produce an amplicondiagnostic for event MZDT09Y. In one aspect, the pair of the firstpolynucleotide primer or a sequence of the second polynucleotide primeris chosen from SEQ ID NO: 7, or the complement thereof; or a sequence ofthe first polynucleotide primer is or is complementary to a corn plantgenome sequence flanking the point of insertion of a heterologous DNAsequence inserted into the corn plant genome of event MZDT09Y, and asequence of the second polynucleotide primer is or is complementary tothe heterologous DNA sequence inserted into the genome of event MZDT09Y.In another aspect, the first polynucleotide primer comprises at least 10contiguous nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and the complements thereof;and the second polynucleotide primer comprises at least 10 contiguousnucleotides from SEQ ID NO: 7, or the complements thereof. In anotheraspect, the first polynucleotide primer comprises a nucleotide sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 32, and the complements thereof; and the second polynucleotideprimer comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 33, and thecomplements thereof. In another aspect, the first polynucleotide primerconsists of SEQ ID NO: 12 and the second polynucleotide primer consistsof SEQ ID NO: 13; or the first polynucleotide primer consists of SEQ IDNO: 14 and the second polynucleotide primer consists of SEQ ID NO: 15;or the first polynucleotide primer consists of SEQ ID NO: 32 and thesecond polynucleotide primer consists of SEQ ID NO: 33.

In another embodiment, the present invention encompasses a method ofdetecting the presence of a nucleic acid molecule that is unique toevent MZDT09Y in a sample comprising corn nucleic acids. In one aspect,the method comprises contacting the sample with a pair of primers that,when used in a nucleic-acid amplification reaction with genomic DNA fromevent MZDT09Y produces an amplicon that is diagnostic for event MZDT09Y;performing a nucleic acid amplification reaction, thereby producing theamplicon; and detecting the amplicon. In another aspect, the methodcomprises contacting the sample with a probe that hybridizes under highstringency conditions with genomic DNA from event MZDT09Y and does nothybridize under high stringency conditions with DNA of a control cornplant; subjecting the sample and probe to high stringency hybridizationconditions; and detecting hybridization of the probe to the nucleic acidmolecule.

In another embodiment, the present invention encompasses a kit fordetecting nucleic acids that are unique to event MZDT09Y comprising atleast one nucleic acid molecule of sufficient length of contiguouspolynucleotides to function as a primer or probe in a nucleic aciddetection method, and which upon amplification of or hybridization to atarget nucleic acid sequence in a sample followed by detection of theamplicon or hybridization to the target sequence, are diagnostic for thepresence of nucleic acid sequences unique to event MZDT09Y in thesample. In one aspect, the nucleic acid molecule of sufficient length ofcontinuous polynucleotides comprises a nucleotide sequence selected fromthe group consisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 8; and the complements thereof. In another aspect, thenucleic acid molecule is selected from the group consisting of SEQ IDNOs: 12-15, SEQ ID NOs: 32-34, and the complements thereof.

In another embodiment, the present invention encompasses a transgeniccorn plant, or cells or tissues thereof, comprising a nucleic acidmolecule that is unique to corn event MZDT09Y. The nucleotide sequencemay comprise any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, or any of the complements thereof. In another embodiment, thepresent invention encompasses a corn seed comprising a nucleic acidmolecule that is unique to corn event MZDT09Y. An example of the seed isdeposited at the American Type Culture Collection under the accessionnumber PTA-13025.

In another embodiment, the present invention encompasses a biologicalsample derived from an event MZDT09Y corn plant, tissue, or seed,wherein the sample comprises a nucleotide sequence which is or iscomplementary to a nucleotide sequence selected from the groupconsisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, andthe complements thereof; and wherein the sequence is detectable in thesample using a nucleic acid amplification or nucleic acid hybridizationmethod. In one aspect, the sample is selected from the group consistingof corn flour, corn meal, corn syrup, corn oil, corn starch, and cerealsmanufactured in whole or in part to contain corn by-products. In anotherembodiment, the present invention encompasses an extract derived fromthe biological sample. In one aspect, the extract is selected from thegroup consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain cornby-products.

In another embodiment, the present invention encompasses a method forproducing a corn plant with increased yield, the method comprisingsexually crossing a first parent corn plant with a second parent cornplant, wherein said first or second parent corn plant comprises eventMZDT09Y DNA, thereby producing a plurality of first generation progenyplants; selecting a first generation progeny plant with increased yield;selfing the first generation progeny plant, thereby producing aplurality of second generation progeny plants; and selecting from thesecond generation progeny plants, a plant with increased yield; whereinthe second generation progeny plants comprise a nucleic acid moleculeselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, and the complements thereof. In one aspect, theincreased yield is indicated by an increase as compared to a controlplant of any one of the following: increased grain yield, increasedseed, increased seed weight, increased biomass, increased sugar,increased oil, increased plant vigor, increased yield under non-optimalconditions, increased yield under stress conditions and increased yieldunder water stress conditions.

In another embodiment, the present invention encompasses a method forproducing a corn plant with increased tolerance to abiotic stress, themethod comprising sexually crossing a first parent corn plant with asecond parent corn plant, wherein said first or second parent corn plantcomprises event MZDT09Y DNA, thereby producing a plurality of firstgeneration progeny plants; selecting a first generation progeny plantwith increased tolerance to abiotic stress; selfing the first generationprogeny plant, thereby producing a plurality of second generationprogeny plants; and selecting from the second generation progeny plants,a plant with increase tolerance to abiotic stress; wherein the secondgeneration progeny plants comprise a nucleic acid molecule selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, and the complements thereof. In one aspect, the abiotic stresscomprises stress selected from the group consisting of water stress,heat stress or cold stress. In another aspect, the water stress iscaused by drought.

In another embodiment, the present invention encompasses a method ofproducing hybrid corn seeds comprising planting seeds of a first inbredcorn line comprising event MZDT09Y and seeds of a second inbred linehaving a genotype different from the first inbred corn line; cultivatingcorn plants resulting from the planting until time of flowering;emasculating the flowers of plants of one of the corn inbred lines;sexually crossing the two different inbred lines with each other;harvesting the hybrid seed produced thereby. The hybrid seed produced bythis method comprise a nucleic acid molecule selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,and the complements thereof. In one aspect, the plants of the firstinbred corn line are the female parents or male parents. In anotherembodiment, the present invention encompasses hybrid seed produced bythe above method of producing hybrid seed.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingsequences, which form a part hereof and in which there is shown by wayof illustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the embodiments recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the 20 by 5′- junction sequence.

SEQ ID NO: 2 is the 20 by 3′- junction sequence.

SEQ ID NO: 3 is the 60 by 5′- genome plus insert sequence.

SEQ ID NO: 4 is the 60 by 3′- insert plus genome sequence.

SEQ ID NO: 5 is the 5′- flanking genomic sequence.

SEQ ID NO: 6 is the 3′- flanking genomic sequence.

SEQ ID NO: 7 is the heterologous insert sequence.

SEQ ID NO: 8 is the heterologous insert sequence plus genomic flankingsequences.

SEQ ID NO: 9 is the OsMADS6 promoter.

SEQ ID NO: 10 is the sequence of the modified T6PP gene.

SEQ ID NO: 11 is the sequence of the modified T6PP protein.

SEQ ID NOs: 12-22 are primers useful in the present invention.

SEQ ID NOs: 23-28 are amplicons useful in the present invention.

SEQ ID NO: 29 is the unmodified rice T6PP (OsT6PP) cDNA sequence.

SEQ ID NO: 30 is the binary construct 15777.

SEQ ID NO: 31 is the binary construct 15769.

SEQ ID NOs: 32-34 are TaqMan primers and probe

SEQ ID NO: 35 is the amplicon produced by the TaqMan PCR reaction.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry, plant quantitativegenetics, statistics and recombinant DNA technology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., Langenheim and Thimann, (1982) Botany: PlantBiology and Its Relation to Human Affairs, John Wiley; Cell Culture andSomatic Cell Genetics of Plants, vol. 1, Vasil, ed. (1984); Stanier, etal., (1986) The Microbial World, 5th ed., Prentice-Hall; Dhringra andSinclair, (1985) Basic Plant Pathology Methods, CRC Press; Maniatis, etal., (1982) Molecular Cloning: A Laboratory Manual; DNA Cloning, vols. Iand II, Glover, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984);Nucleic Acid Hybridization, Hames and Higgins, eds. (1984); and theseries Methods in Enzymology, Colowick and Kaplan, eds, Academic Press,Inc., San Diego, Calif.

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, plant species or genera,constructs, and reagents described as such. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention.

As used herein the singular forms “a”, “and”, and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a vector” is a reference to one or more vectorsand includes equivalents thereof known to those skilled in the art.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 percent.

As used herein, the word “or” means any one member of a particular listand also includes any combination of members on that list.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. The term“consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between. As used herein the term“method” refers to manners, means, techniques and procedures foraccomplishing a given task including, but not limited to, those manners,means, techniques and procedures either known to, or readily developedfrom known manners, means, techniques and procedures by practitioners ofthe chemical, pharmacological, biological, biochemical and medical arts.It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

By “microbe” is meant any microorganism (including both eukaryotic andprokaryotic microorganisms), such as fungi, yeast, bacteria,actinomycetes, algae and protozoa, as well as other unicellularstructures.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), O-Beta Replicasesystems, transcription-based amplification system (TAS) and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Persing, et al., eds.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidsthat encode identical or conservatively modified variants of the aminoacid sequences. Because of the degeneracy of the genetic code, a largenumber of functionally identical nucleic acids encode any given protein.For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinthat encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; one exception is Micrococcus rubens, for which GTGis the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol.139:425-32) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid, which encodes apolypeptide of the present invention, is implicit in each describedpolypeptide sequence and incorporated herein by reference.

A “control plant” or “control” as used herein may be a non-transgenicplant of the parental line used to generate a transgenic plant herein. Acontrol plant may in some cases be a transgenic plant line that includesan empty vector or marker gene, but does not contain the recombinantpolynucleotide of the present invention that is expressed in thetransgenic plant being evaluated. A control plant in other cases is atransgenic plant expressing the gene with a constitutive promoter. Ingeneral, a control plant is a plant of the same line or variety as thetransgenic plant being tested, lacking the specific trait-conferring,recombinant DNA that characterizes the transgenic plant. Such aprogenitor plant that lacks that specific trait-conferring recombinantDNA can be a natural, wild-type plant, an elite, non-transgenic plant,or a transgenic plant without the specific trait-conferring, recombinantDNA that characterizes the transgenic plant. The progenitor plantlacking the specific, trait-conferring recombinant DNA can be a siblingof a transgenic plant having the specific, trait-conferring recombinantDNA. Such a progenitor sibling plant may include other recombinant DNA

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” when the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%,preferably 60-90% of the native protein for its native substrate.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton, Proteins, W. H. Freeman and Co. (1984).

As used herein the terms “modified” or “modification” interchangeablyrefer to deliberate or random substitutions, deletions or additions to anucleic acid, peptide, polypeptide or protein sequence which alters,adds or deletes at least one amino acid residue within a givenpolypeptide. A “modified T6PP” as used herein refers to any nucleic acidencoding a T6PP or peptides, polypeptides or protein having T6PPactivity either of which having been modified so that the resultant T6PPconfers decreased T6PP activity and/or decreased binding to T6P.

By “encoding” or “encoded,” with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal and fungalmitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al.,(1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliateMacronucleus, may be used when the nucleic acid is expressed using theseorganisms.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledonous plants or dicotyledonous plants as thesepreferences have been shown to differ (Murray, et al., (1989) NucleicAcids Res. 17:477-98 and herein incorporated by reference). Thus, themaize preferred codon for a particular amino acid might be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra.

As used herein, a “heterologous” nucleic acid sequence is a nucleic acidsequence not naturally associated with a host cell into which it isintroduced, including non-naturally occurring multiple copies of anaturally occurring nucleic acid sequence. A heterologous nucleic acidrefers to a nucleic acid that originates from a foreign species withrespect to a host cell or from the same species as the host cell,provided the heterologous nucleic acid sequence is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. A heterologous protein may originate froma foreign species, or if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which comprises a heterologous nucleicacid sequence of the invention, which comprises an expression cassetteand supports the replication and/or expression of the expressioncassette. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, plant, amphibian or mammaliancells. Preferably, host cells are monocotyledonous or dicotyledonousplant cells, including but not limited to maize, sorghum, sunflower,soybean, wheat, alfalfa, rice, cotton, canola, barley, millet andtomato. A particularly preferred monocotyledonous host cell is a maizehost cell.

As used herein, the term transgenic “event” refers to a recombinantplant produced by transformation and regeneration of a plant cell ortissue with heterologous DNA, for example, an expression cassette thatincludes a gene of interest. The term “event” refers to the originaltransformant and/or progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another corn line. Evenafter repeated backcrossing to a recurrent parent, the inserted DNA andthe flanking DNA from the transformed parent is present in the progenyof the cross at the same chromosomal location. The term “event” alsorefers to DNA from the original transformant comprising the inserted DNAand flanking genomic sequence immediately adjacent to the inserted DNAthat would be expected to be transferred to a progeny that receivesinserted DNA including the transgene of interest as the result of asexual cross of one parental line that includes the inserted DNA (e.g.,the original transformant and progeny resulting from selfing) and aparental line that does not contain the inserted DNA. Normally,transformation of plant tissue produces multiple events, each of whichrepresent insertion of a DNA construct into a different location in thegenome of a plant cell. Based on the expression of the transgene orother desirable characteristics, a particular event is selected. Thus,“event MZDT09Y”, “MZDT09Y”, “09Y” or “09Y event” may be usedinterchangeably.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon ortransiently expressed (e.g., transfected mRNA).

As used herein “gene stack” refers to the introduction of two or moregenes into the genome of an organism. In certain aspects of theinvention it may be desirable to stack any abiotic stress gene (e.g.cold shock proteins, genes associated with ABA response) with the T6PPsas described herein. Likewise, it may also be desirable to stack theT6PPs as described herein with genes conferring insect resistance,disease resistance, increased yield or any other beneficial trait (e.g.increased plant height, etc.) known in the art.

The terms “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment. Incontrast, a non-isolated nucleic acid, such as DNA or RNA, is found inthe state in which it exists in nature. An isolated nucleic acid may bein a transgenic plant and still be considered “isolated.” Nucleic acids,which are “isolated,” as defined herein, are also referred to as“heterologous” nucleic acids.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules, which comprise in one case a substantial representation ofthe entire transcribed fraction of a genome of a specified organism.Construction of exemplary nucleic acid libraries, such as genomic andcDNA libraries, is taught in standard molecular biology references suchas Berger and Kimmel, (1987) Guide To Molecular Cloning Techniques, fromthe series Methods in Enzymology, vol. 152, Academic Press, Inc., SanDiego, Calif.; Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual, 2nd ed., vols. 1-3; and Current Protocols in Molecular Biology,Ausubel, et al., eds, Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1994Supplement). In another instance “nucleic acid library” as definedherein may also be understood to represent libraries comprising aprescribed faction or rather not substantially representing an entiregenome of a specified organism. For example, small RNAs, mRNAs andmethylated DNA. A nucleic acid library as defined herein might alsoencompass variants of a particular molecule (e.g. a collection ofvariants for a particular protein).

As used herein “operably linked” includes reference to a functionallinkage between a first sequence, such as a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs, tissues (e.g., leaves, stems, roots, etc.), seeds andplant cells and progeny of same. Plant cell, as used herein includes,without limitation, seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen and microspores. The class of plants, which can beused in the methods of the invention, is generally as broad as the classof higher plants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants including species from thegenera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium and Triticum. Aparticularly preferred plant is Zea mays.

As used herein, “yield” may include reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically formaize, for example), and the volume of biomass generated (for foragecrops such as alfalfa and plant root size for multiple crops). Grainmoisture is measured in the grain at harvest. The adjusted test weightof grain is determined to be the weight in pounds per bushel, adjustedfor grain moisture level at harvest. Biomass is measured as the weightof harvestable plant material generated. Yield can be affected by manyproperties including without limitation, plant height, pod number, podposition on the plant, number of internodes, incidence of pod shatter,grain size, efficiency of nodulation and nitrogen fixation, efficiencyof nutrient assimilation, carbon assimilation, plant architecture,percent seed germination, seedling vigor, and juvenile traits. Yield canalso be affected by efficiency of germination (including germination instressed conditions), growth rate (including growth rate in stressedconditions), ear number, seed number per ear, seed size, composition ofseed (starch, oil, protein) and characteristics of seed fill. Yield of aplant of the can be measured in a number of ways, including test weight,seed number per plant, seed weight, seed number per unit area (i.e.seeds, or weight of seeds, per acre), bushels per acre, tons per acre,or kilo per hectare. For example, corn yield may be measured asproduction of shelled corn kernels per unit of production area, forexample in bushels per acre or metric tons per hectare, often reportedon a moisture adjusted basis, for example at 15.5 percent moisture.Moreover a bushel of corn is defined by law in the State of Iowa as 56pounds by weight, a useful conversion factor for corn yield is: 100bushels per acre is equivalent to 6.272 metric tons per hectare. Othermeasurements for yield are common practice in the art In certainembodiments of the invention yield may be increased in stressed and/ornon-stressed conditions.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or analogs thereof that havethe essential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses and bacteria which comprisegenes expressed in plant cells such Agrobacterium or Rhizobium. Examplesare promoters that preferentially initiate transcription in certaintissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheidsor sclerenchyma. Such promoters are referred to as “tissue preferred.” A“cell type” specific promoter primarily drives expression in certaincell types in one or more organs, for example, vascular cells in rootsor leaves. An “inducible” or “regulatable” promoter is a promoter, whichis under environmental control. Examples of environmental conditionsthat may affect transcription by inducible promoters include anaerobicconditions or the presence of light. Another type of promoter is adevelopmentally regulated promoter, for example, a promoter that drivesexpression during pollen development. Tissue preferred, cell typespecific, developmentally regulated and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter, which is active under most environmental conditions in mostcells.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention. According to some embodiments of theinvention, the promoter is a constitutive promoter, a tissue-specific,or an abiotic stress-inducible promoter.

Suitable constitutive promoters include, for example, CaMV 35S promoter(SEQ ID NO:1546; Odell et al., Nature 313:810-812, 1985); ArabidopsisAt6669 promoter (SEQ ID NO:1652; see PCT Publication No. W004081173A2);maize Ubi 1 (Christensen et al., Plant Mol. Biol. 18:675-689, 1992);rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last etal., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al.,Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant JNovember; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol.Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al., Plant MolBiol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant J. 10(1);107-121,1996), constitutive root tip CT2 promoter (SEQ ID NO:1535; see also PCTapplication No. IL/2005/000627) and Synthetic Super MAS (Ni et al., ThePlant Journal 7: 661-76, 1995). Other constitutive promoters includethose in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121;5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters [such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specificgenes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al.,J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol.14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol.18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214,1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22,1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke etal., Plant Mol Biol, 143).323-32 1990), napA (Stalberg, et al., Planta199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184,1997), sunflower oleosin (Cummins, etal., Plant Mol. Biol. 19: 873-876,1992)], endosperm specific promoters [e.g., wheat LMW and HMW,glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b andg gliadins (EMBO3:1409-15, 1984), Barley ltrl promoter, barley B1, C, Dhordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; MolGen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal,116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter(Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolaminNRP33, rice -globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8)885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol.Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68,1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgumgamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], embryo specificpromoters [e.g., rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93:8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71,1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], andflower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Vander Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell etal., Mol. Gen Genet. 217:240-245; 1989), apetala-3].

Suitable abiotic stress-inducible promoters include, but not limited to,salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al.,Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such asmaize rabl7 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266,1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295,1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol. Biol.39:373-380, 1999); heat-inducible promoters such as heat tomatohsp80-promoter from tomato (U.S. Pat. No. 5,187,267).

The term “Enzymatic activity” is meant to include demethylation,hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, andO-dealkylations, desulfation, deamination, and reduction of azo, nitro,and N-oxide groups. The term “nucleic acid” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, or sense or anti-sense, and unless otherwiselimited, encompasses known analogues of natural nucleotides thathybridize to nucleic acids in a manner similar to naturally occurringnucleotides. Unless otherwise indicated, a particular nucleic acidsequence includes the complementary sequence thereof.

A “structural gene” is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding the5′ sequence which drives the initiation of transcription. The structuralgene may alternatively encode a nontranslatable product. The structuralgene may be one which is normally found in the cell or one which is notnormally found in the cell or cellular location wherein it isintroduced, in which case it is termed a “heterologous gene”. Aheterologous gene may be derived in whole or in part from any sourceknown to the art, including a bacterial genome or episome, eukaryotic,nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. Astructural gene may contain one or more modifications that could affectbiological activity or its characteristics, the biological activity orthe chemical structure of the expression product, the rate of expressionor the manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions and substitutions ofone or more nucleotides. The structural gene may constitute anuninterrupted coding sequence or it may include one or more introns,bounded by the appropriate splice junctions. The structural gene may betranslatable or non-translatable, including in an anti-senseorientation. The structural gene may be a composite of segments derivedfrom a plurality of sources and from a plurality of gene sequences(naturally occurring or synthetic, where synthetic refers to DNA that ischemically synthesized).

“Derived from” is used to mean taken, obtained, received, traced,replicated or descended from a source (chemical and/or biological). Aderivative may be produced by chemical or biological manipulation(including, but not limited to, substitution, addition, insertion,deletion, extraction, isolation, mutation and replication) of theoriginal source.

“Chemically synthesized”, as related to a sequence of DNA, means thatportions of the component nucleotides were assembled in vitro. Manualchemical synthesis of DNA may be accomplished using well establishedprocedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983),Weissman (ed.), Praeger Publishers, New York, Chapter 1); automatedchemical synthesis can be performed using one of a number ofcommercially available machines.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention or mayhave reduced or eliminated expression of a native gene. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, an “expression cassette” is a nucleic acid construct,generated recombinantly or synthetically, with a series of specifiednucleic acid elements, which permit transcription of a particularnucleic acid in a target cell. The expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA,virus or nucleic acid fragment. Typically, the expression cassetteportion of an expression vector includes, among other sequences, anucleic acid to be transcribed and a promoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 40% sequence identity, preferably 60-90% sequenceidentity and most preferably 100% sequence identity (i.e.,complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which can be up to 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Optimally, the probe is approximately 500 nucleotides inlength, but can vary greatly in length from less than 500 nucleotides toequal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide or Denhardt's.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C. and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C. and a wash in 0.1×SSC at 60 to 65° C. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-84:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)⁻0.61 (% form)-−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, New York (1993); and Current Protocols inMolecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995). Unless otherwise stated, in thepresent application high stringency is defined as hybridization in4×SSC, 5×Denhardt′s (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovineserum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA,and 25 mM Na phosphate at 65° C. and a wash in 0.1×SSC, 0.1% SDS at 65°C.

As used herein, “transgenic plant” includes reference to a plant, whichcomprises within its genome a heterologous nucleic acid sequence.Generally, the heterologous nucleic acid sequence is stably integratedwithin the genome such that the nucleic acid sequence is passed on tosuccessive generations. The heterologous nucleic acid sequence may beintegrated into the genome alone or as part of a recombinant expressioncassette. “Transgenic” is used herein to include any cell, cell line,callus, tissue, plant part or plant, the genotype of which has beenaltered by the presence of a heterologous nucleic acid sequence,including those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transpositionor spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

“Overexpression” refers to the level of expression in transgenicorganisms that exceeds levels of expression in normal or untransformedorganisms.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

“Preferred expression”, “Preferential transcription” or “preferredtranscription” interchangeably refers to the expression of gene productsthat are preferably expressed at a higher level in one or a few planttissues (spatial limitation) and/or to one or a few plant developmentalstages (temporal limitation) while in other tissues/developmental stagesthere is a relatively low level of expression.

“Primary transformant” and “TO generation” refer to transgenic plantsthat are of the same genetic generation as the tissue that was initiallytransformed (i.e., not having gone through meiosis and fertilizationsince transformation). “Secondary transformants” and the “T1, T2, T3,etc. generations” refer to transgenic plants derived from primarytransformants through one or more meiotic and fertilization cycles. Theymay be derived by self-fertilization of primary or secondarytransformants or crosses of primary or secondary transformants withother transformed or untransformed plants.

A “selectable marker gene” refers to a gene whose expression in a plantcell gives the cell a selective advantage. The selective advantagepossessed by the cells transformed with the selectable marker gene maybe due to their ability to grow in presence of a negative selectiveagent, such as an antibiotic or a herbicide, compared to the ability togrow of non-transformed cells. The selective advantage possessed by thetransformed cells may also be due to their enhanced capacity, relativeto non-transformed cells, to utilize an added compound as a nutrient,growth factor or energy source. A selective advantage possessed by atransformed cell may also be due to the loss of a previously possessedgene in what is called “negative selection”. In this, a compound isadded that is toxic only to cells that did not lose a specific gene (anegative selectable marker gene) present in the parent cell (typically atransgene).

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. “Transiently transformed” refers to cells in whichtransgenes and foreign DNA have been introduced (for example, by suchmethods as Agrobacterium-mediated transformation or biolisticbombardment), but not selected for stable maintenance. “Stablytransformed” refers to cells that have been selected and regenerated ona selection media following transformation.

“Transformed,” “transgenic,” and “recombinant” are used interchangeablyand each refer to a host organism such as a bacterium or a plant intowhich a heterologous nucleic acid molecule has been introduced. Thenucleic acid molecule can be stably integrated into the genome of thehost or the nucleic acid molecule can also be present as anextrachromosomal molecule. Such an extrachromosomal molecule can beauto-replicating. Transformed cells, tissues, or plants are understoodto encompass not only the end product of a transformation process, butalso transgenic progeny thereof. A “non-transformed”, “non-transgenic”,or “non-recombinant” host refers to a wild-type organism, e.g., abacterium or plant, which does not contain the heterologous nucleic acidmolecule.

The term “translational enhancer sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translational enhancer sequencemay affect processing of the primary transcript to mRNA, mRNA stabilityor translation efficiency. “Visible marker” refers to a gene whoseexpression does not confer an advantage to a transformed cell but can bemade detectable or visible. Examples of visible markers include but arenot limited to β-glucuronidase (GUS), luciferase (LUC) and greenfluorescent protein (GFP).

“Wild-type” refers to the normal gene, virus, or organism found innature without any mutation or modification.

As used herein, “plant material,” “plant part” or “plant tissue” meansplant cells, plant protoplasts, plant cell tissue cultures from whichplants can be regenerated, plant calli, plant clumps, and plant cellsthat are intact in plants or parts of plants such as embryos, pollen,ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs,husks, stalks, roots, root tips, anthers, tubers, rhizomes and the like.

As used herein “Protein extract” refers to partial or total proteinextracted from a plant part. Plant protein extraction methods are wellknown in the art.

As used herein “plant sample” or “biological sample” refers to eitherintact or non-intact (e g milled seed or plant tissue, chopped planttissue, lyophilized tissue) plant tissue. It may also be an extractcomprising intact or non-intact seed or plant tissue. The biologicalsample or extract may be selected from the group consisting of cornflour, corn meal, corn syrup, corn oil, corn starch, and cerealsmanufactured in whole or in part to contain corn by-products.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides or polypeptides:(a) “reference sequence,” (b) “comparison window,” (c) “sequenceidentity,” (d) “percentage of sequence identity” and (e) “substantialidentity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, and 100 or longer. Those of skill in the art understand thatto avoid a high similarity to a reference sequence due to inclusion ofgaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, mayconduct optimal alignment of sequences for comparison; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG® programs (Accelrys, Inc., San Diego, Calif.).). The CLUSTALprogram is well described by Higgins and Sharp, (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. The preferred program to use for optimal global alignment ofmultiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol.,25:351-60 which is similar to the method described by Higgins and Sharp,(1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLASTfamily of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., eds., GreenePublishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the Wisconsin Genetics Software Package are 8 and 2, respectively.The gap creation and gap extension penalties can be expressed as aninteger selected from the group of integers consisting of from 0 to 100.Thus, for example, the gap creation and gap extension penalties can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 and 50 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has between 50-100% sequenceidentity, preferably at least 50% sequence identity, preferably at least60% sequence identity, preferably at least 70%, more preferably at least80%, more preferably at least 90% and most preferably at least 95%,compared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of between 55-100%,preferably at least 55%, preferably at least 60%, more preferably atleast 70%, 80%, 90% and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.The degeneracy of the genetic code allows for many amino acidssubstitutions that lead to variety in the nucleotide sequence that codefor the same amino acid, hence it is possible that the DNA sequencecould code for the same polypeptide but not hybridize to each otherunder stringent conditions. This may occur, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide, which the first nucleicacid encodes, is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The phrase “abiotic stress” as used herein refers to any adverse effecton metabolism, growth, reproduction and/or viability of a plant byabiotic factors (i.e. water availability, heat, cold, and etc).Accordingly, abiotic stress can be induced by suboptimal environmentalgrowth conditions such as, for example, salinity, water deprivation,water deficit, drought, flooding, freezing, low or high temperature (e g, chilling or excessive heat), toxic chemical pollution, heavy metaltoxicity, anaerobiosis, nutrient deficiency, nutrient excess,atmospheric pollution or UV irradiation.

The phrase “abiotic stress tolerance” as used herein refers to theability of a plant to endure an abiotic stress without suffering asubstantial alteration in metabolism, growth, productivity and/orviability.

As used herein “water deficit” means a period when water available to aplant is not replenished at the rate at which it is consumed by theplant. A long period of water deficit is colloquially called drought.Lack of rain or irrigation may not produce immediate water stress ifthere is an available reservoir of ground water to support the growthrate of plants. Plants grown in soil with ample groundwater can survivedays without rain or irrigation without adverse effects on yield. Plantsgrown in dry soil are likely to suffer adverse effects with minimalperiods of water deficit. Severe water deficit stress can cause wilt andplant death; moderate drought can reduce yield, stunt growth or retarddevelopment. Plants can recover from some periods of water deficitstress without significantly affecting yield. However, water deficit atthe time of pollination can lower or reduce yield. Thus, a useful periodin the life cycle of corn, for example, for observing response ortolerance to water deficit is the late vegetative stage of growth beforetassel emergence or the transition to reproductive development.Tolerance to water deficit is determined by comparison to controlplants. For instance, plants of this invention can produce a higheryield than control plants when exposed to water deficit. In thelaboratory and in field trials drought can be simulated by giving plantsof this invention and control plants less water than is given tosufficiently-watered control plants and measuring differences in traits.One aspect of the invention provides plants overexpressing the genes asdisclosed herein which confers a higher tolerance to a water deficit.

As used herein, the phrase “water optimization” refers to any measure ofa plant, its parts, or its structure that can be measured and/orquantified in order to assess an extent of or a rate of plant growth anddevelopment under different conditions of water availability. As such, a“water optimization trait” is any trait that can be shown to influenceyield in a plant under different sets of growth conditions related towater availability. Exemplary measures of water optimization are grainyield at standard moisture percentage (YGSMN), grain moisture at harvest(GMSTP), grain weight per plot (GWTPN), and percent yield recovery(PYREC).

As used herein, the phrases “drought tolerance” and “drought tolerant”refer to a plant's ability to endure and/or thrive under conditionswhere water availability is suboptimal. . In general, a plant is labeledas “drought tolerant” if it displays “enhanced drought tolerance.” Asused herein, the phrase “enhanced drought tolerance” refers to ameasurable improvement, enhancement, or increase in one or more wateroptimization phenotypes as compared to one or more control plants.

Water Use Efficiency (WUE) is a parameter frequently used to estimatethe tradeoff between water consumption and CO₂ uptake/growth (Kramer,1983, Water Relations of Plants, Academic Press p. 405). WUE has beendefined and measured in multiple ways. One approach is to calculate theratio of whole plant dry weight, to the weight of water consumed by theplant throughout its life (Chu et al., 1992, Oecologia 89:580). Anothervariation is to use a shorter time interval when biomass accumulationand water use are measured (Mian et al., 1998, Crop Sci. 38:390).Another approach is to utilize measurements from restricted parts of theplant, for example, measuring only aerial growth and water use (Nienhuiset al 1994 Amer J Bot 81:943). WUE also has been defined as the ratio ofCO₂ uptake to water vapor loss from a leaf or portion of a leaf, oftenmeasured over a very short time period (e.g. seconds/minutes) (Kramer,1983, p. 406). The ratio of ¹³C/ ¹²C fixed in plant tissue, and measuredwith an isotope ratio mass-spectrometer, also has been used to estimateWUE in plants using C-3 photosynthesis (Martin et al., 1999, Crop Sci.1775). As used herein, the term “water use efficiency” refers to theamount of organic matter produced by a plant divided by the amount ofwater used by the plant in producing it, i.e. the dry weight of a plantin relation to the plant's water use. As used herein, the term “dryweight” refers to everything in the plant other than water, andincludes, for example, carbohydrates, proteins, oils, and mineralnutrients. It is contemplated that the transgenic plants produced by themethods described herein will confer an increase in water useefficiency.

The phrase “biotic stress” as used herein refers to any adverse effecton metabolism, growth, reproduction and/or viability of a plant bybiotic factors (i.e. insect pressure, disease and etc).

The phrase “biotic stress tolerance” as used herein refers to theability of a plant to endure an biotic stress without suffering asubstantial alteration in metabolism, growth, reproduction and/orviability.

As used herein the phrase “plant biomass” refers to the amount (measuredin grams of air-dry or dry tissue) of a tissue produced from the plantin a growing season, which could also determine or affect the plantyield or the yield per growing area.

As used herein the phrase “plant vigor” refers to the amount (measuredby weight) of tissue produced by the plant in a given time. Henceincreased vigor could determine or affect the plant yield or the yieldper growing time or growing area.

The term “early vigor” refers to active healthy well-balanced growthespecially during early stages of plant growth, and may result fromincreased plant fitness due to, for example, the plants being betteradapted to their environment (optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigor alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (e.g. crops growing in auniform fashion, such as the crops reaching various stages ofdevelopment at substantially the same time), and often higher yields.Therefore, early vigor may be determined by measuring various factors,such as thousand kernel weight, percentage germination, percentageemergence, seedling growth, seedling height, root length, root and shootbiomass and many more.

As used herein, “seedling vigor” refers to the plant characteristicwhereby the plant emerges from soil faster, has an increased germinationrate (i.e., germinates faster), has faster and larger seedling growthand/or germinates faster under cold conditions as compared to the wildtype or control under similar conditions. Seedling vigor has often beendefined to comprise the seed properties that determine “the potentialfor rapid, uniform emergence and development of normal seedlings under awide range of field conditions”.

The life cycle of flowering plants in general can be divided into threegrowth phases: vegetative, inflorescence, and floral (late inflorescencephase). In the vegetative phase, the shoot apical meristem (SAM)generates leaves that later will ensure the resources necessary toproduce fertile offspring. Upon receiving the appropriate environmentaland developmental signals the plant switches to floral, or reproductive,growth and the SAM enters the inflorescence phase (I) and gives rise toan inflorescence with flower primordia. During this phase the fate ofthe SAM and the secondary shoots that arise in the axils of the leavesis determined by a set of meristem identity genes, some of which preventand some of which promote the development of floral meristems. Onceestablished, the plant enters the late inflorescence phase where thefloral organs are produced. If the appropriate environmental anddevelopmental signals are present the plant switches to floral, orreproductive, growth. If such signals are disrupted, the plant will notbe able to enter reproductive growth, therefore maintaining vegetativegrowth.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells, which can be cultured into a whole plant.

Plants engineered for improved yield under various biotic and abioticstresses is of special interest in the field of agriculture. Forexample, abiotic stress is a primary cause of crop loss worldwide,reducing average yields for most major crop plants by more than 50%(Wang et al., Planta (2003) 218: 1-14). Abiotic stresses may be causedby drought, floods, salinity, extremes of temperature, chemical toxicityand oxidative stress. The ability to improve plant tolerance to abioticstress would be of great economic advantage to farmers worldwide andwould allow for the cultivation of crops during adverse conditions andin territories where cultivation of crops may not otherwise be possible.

In some instances plant yield is relative to the amount of plant biomassa particular plant may produce. A larger plant with a greater leaf areacan typically absorb more light, nutrients and carbon dioxide than asmaller plant and therefore will likely gain a greater weight during thesame period (Fasoula & Tollenaar 2005 Maydica 50:39). Increased plantbiomass may also be highly desirable in processes such as the conversionof biomass (e.g. corn, grasses, sorghum, cane) to fuels such as forexample ethanol or butanol.

The ability to increase plant yield would have many applications inareas such as agriculture, the production of ornamental plants,arboriculture, horticulture, biofuel production, pharmaceuticals, enzymeindustries which use plants as factories for these molecules andforestry. Increasing yield may also find use in the production ofmicrobes or algae for use in bioreactors (for the biotechnologicalproduction of substances such as pharmaceuticals, antibodies, vaccines,fuel or for the bioconversion of organic waste) and other such areas.

Plant breeders are often interested in improving specific aspects ofyield depending on the crop or plant in question, and the part of thatplant or crop which is of relative economic value. For example, a plantbreeder may look specifically for improvements in plant biomass (weight)of one or more parts of a plant, which may include aboveground(harvestable) parts and/or harvestable parts below ground. This isparticularly relevant where the aboveground parts or below ground partsof a plant are for consumption. For many crops, particularly cereals, animprovement in seed yield is highly desirable. Increased seed yield maymanifest itself in many ways with each individual aspect of seed yieldbeing of varying importance to a plant breeder depending on the crop orplant in question and its end use.

It would be of great advantage to a plant breeder to be able to pick andchoose the aspects of yield to be altered. It may also be highlydesirable to be able to pick a gene suitable for altering a particularaspect of yield (e.g. seed yield, biomass weight, water use efficiency,yield under stress conditions). For example an increase in the fillrate, combined with increased thousand kernel weight would be highlydesirable for a crop such as corn. For rice and wheat a combination ofincreased fill rate, harvest index and increased thousand kernel weightwould be highly desirable.

It has now been discovered that the expression of several forms oftrehalose-6-phosphate phosphatase (T6PP) in plants confers a significantincrease in yield as well as confer resistance to various types ofstress (i.e. abiotic stress). During the course of analyzing variousT6PP variants transgenically expressed in plants it has been found thatthe transgenic plants showing the highest yield in seed also comprisedT6PPs with modifications to amino acid residues associated withsubstrate binding. Not to be limited by theory, these proteins may havedecreased activity as compared to a T6PP not containing thesemodifications. Further, not to be limited by theory, it appears thatexpressing a T6PP in plant with decreased activity results in abeneficial phenotype having significant increased yield in both stress(e.g. drought) and non-stressed field conditions. T6PP is an enzymeinvolved in the trehalose biosynthesis pathway. Trehalose, anon-reducing disaccharide consisting of two glucose molecules linked viaalpha-1,1 bonds. The sugar trehalose can be found in many variousorganisms across multiple kingdoms (e.g. plants, bacteria, insects,etc). Trehalose has been shown to be involved in carbohydrate storagefunction and has been further associated to play a role in stresstolerance in bacteria, fungi and insects. In plants, trehalose wasinitially thought to be confined to extremophiles such as theresurrection plant Selaginella lepidophylla, however it is now widelyaccepted that trehalose metabolism is ubiquitous in the plant kingdom.

Trehalose is synthesized from UDP-glucose and Glucose-6-phosphate in twoenzymatic reactions. First UDP-glucose and Glucose-6-phosphate areconverted to UDP (uridine diphosphate) and alpha, alpha-trehalose6-phosphate (T6P) by the enzyme T6PS (trehalose phosphate synthase). Ina second step, which is catalyzed by the enzyme T6PP (trehalosephosphate phosphatase), T6P is de-phosphorylated to produce trehaloseand orthophosphate.

In yeast, the two enzymatic activities (T6PS and T6PP activity) residein a large protein complex, containing the active subunits, T6PS 1 andT6PS2, and the regulatory subunits, with T6PS1 having T6PS activity andT6PS2 having T6PP activity. In E. coli, the two enzymatic activities arefound in separate protein complexes. In plants, the protein complex hasnot been characterized to date.

In Arabidopsis thaliana, trehalose biosynthetic enzymes have beenclassified into three classes:

Class I: containing four genes, AtT6PS1 to AtT6PS4 having highsimilarity to ScT6PS1;

Class II: having seven members, AtT6PS5 to AtT6PS1 1, with high sequencesimilarity to ScT6PS2; and

Class III:, containing 10 members, AtT6PPA to AtT6PPJ, encoding proteinswith similarity to E. coli T6PS2 and the C-terminus of ScT6PS2 proteins.

Genes encoding proteins within these classes are also present in otherplant species.

Within Class I and Class II, enzymatic activity has only beenunambiguously determined for AtT6PS1, which displays T6PS activity(Blazquez et al. Plant J. March 1998;13(5):685-9.). Surprisingly, noT6PP activity has been reported to date for any of the other Class IIT6PS proteins. In contrast, T6PP activity was previously described forAtT6PPA and AtT6PPB, two of the members of Class III (Vogel et al. PlantJ. March 1998;13(5):673-83). Plant Class III T6PPs contain twophosphatase consensus sequence motifs found in all T6PP enzymesdescribed to date (Thaller et al. Protein Sci. July 1998;7(7):1647-52).

The genetic manipulation of trehalose biosynthesis genes has beenreported to lead to improved stress tolerance in plants, as well ascausing striking developmental alterations. Overexpression of E. coliOtsA and OtsB genes (equivalents to T6PP and T6PS) in transgenic tobaccoand potato plants was reported to cause developmental aberrations inroots and leaves as well as stunted plant growth. Fewer seeds wereproduced in the OtsA transgenic tobacco plants and the OtsB transgenicpotato plants did not produced tubers (Goddijn et al. Plant Physiol.January 1997;113(1):181-90). Similar results have been described byothers (Holmstrom et al. Nature, 379, 683-684; Romero et al. Planta,201, 293-297; Pilont-Smits et al. 1998; J Plant Physiol. 152:525-532;Schluepmann et al. Proc Natl. Acad. Sci. U S A. 2003;100(11):6849-54).Mutants defective in T6PS and T6PP genes have also reportedly showndevelopmental defects. T6PS 1 knock out mutants in Arabidopsis showedimpaired embryo development (Eastmond et al. Plant J. January 2002;29(2):225-35). McSteen et. al. (Plant Cell 2006; 18; 518-522) mentionsthe isolation and characterization of a maize geneRAMOSA3 (RA3) reportedto be responsible for meristem development and inflorescence developmentincluding branching. It is suggested that the gene, gene product, andregulatory regions may be used to manipulate branching, meristem growth,inflorescence development and arrangement. Negative phenotypesassociated with the expression of a transgene can have detrimentaleffects to a plant's relative yield. For example, without seed set, seedfilling, fertility of a plant etc. there would be no increase in seedyield. Patent application U.S. 2007/0006344 is a first account to thisapplication's knowledge of a method which describes the expression of aT6PP in a plant to confer an increase in plant yield without anynegative phenotypes and/or detrimental effects to the plant biologicalfunction. U.S. Patent Application 2007/0006344 describes the use of atrehalose-6-phosphate phosphatase operably linked to a OsMADS promoterthat targets the preferential expression of the T6PP to maternalreproductive tissue of a plant which resulted in a significant yieldincrease in maize under stress and non-stress conditions. The followinginvention generally involves the identification of T6PP havingmodifications that confer improved yield and field efficacy in cropplants and further describes methods one may use to increase yield in aplant by utilizing modified T6PPs in a plant.

As used herein the term “reduced activity” refers to any decrease inT6PP activity.

T6PP proteins (at least in their native form) typically havetrehalose-6-phosphate phosphatase activity. Polypeptides withtrehalose-6-phosphate phosphatase activity belong to the enzymatic classof EC:3.1.3.12, according to the classification of the Enzyme Commissionof the Nomenclature Committee of the International Union of Biochemistryand Molecular Biology (NC-IUBMB). Enzymes in class EC:3.1.3.12 catalyzethe reaction: trehalose-6-phosphate+H₂O=trehalose+phosphate. It iscontemplated that any T6PP or protein having T6PP activity can bemodified for decreased activity via methods such as point mutation(s),irradiation, etc to confer the positive effects as described herein whenexpressed transgenically in a plant.

The activity of a trehalose-6-phosphate phosphatase protein may bemeasured by determining the levels of the substrate processed and thelevels of product accumulated in an in vitro reaction, that is, bydetermining the level of trehalose-6-phosphate consumption and/ortrehalose accumulation from the reaction. Enzymatic methods to measuretrehalose can be based on hydrolyzing trehalose to glucose, such asthose described by Van Dijck et al. Biochem J. August 2002 15;366(Pt1):63-71 and Zentella et al. Plant Physiol. April 1999;119(4):1473-82.

Trehalose-6-phosphate levels may also be measured by HPLC (HighPerformance Liquid Chromatography) methods as described by Avonce et al.Plant Physiol. November 2004; 136(3):3649-59; Schluepmann et al. 2003.Alternative methods based on determining the release of inorganicphosphate from trehalose-6-phosphate have also been described Klutts etal. J Biol Chem. January 2003 24;278(4):2093-100. An alternative methodto determine trehalose-6-phosphate levels using liquid chromatographycoupled to MS-Q3 (triple quadrupole MS) has been described by Lunn etal. Biochem J. July 2006 1;397(1):139-48.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die).

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

The transfer of foreign genes into the genome of a plant, other than bybreeding, is called transformation. Any of several transformationmethods may be used to introduce the gene of interest into a suitableancestor cell. The methods described for the transformation andregeneration of plants from plant tissues or plant cells may be utilizedfor transient or for stable transformation. Transformation methodsinclude the use of liposomes, electroporation, chemicals that increasefree DNA uptake, injection of the DNA directly into the plant, particlegun bombardment, transformation using viruses or pollen andmicroprojection. Methods may be selected from the calcium/polyethyleneglycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296,72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373);electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol3, 1099-1102); microinjection into plant material (Crossway A et al.,(1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particlebombardment (Klein T M et al., (1987) Nature 327: 70) infection with(non-integrative) viruses and the like. Transgenic plants, includingtransgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is thetransformation in planta. To this end, it is possible, for example, toallow the agrobacteria to act on plant seeds or to inoculate the plantmeristem with agrobacteria. It has proved particularly expedient inaccordance with the invention to allow a suspension of transformedagrobacteria to act on the intact plant or at least on the flowerprimordia. The plant is subsequently grown on until the seeds of thetreated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium -mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 Al, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens , for example pBin19 (Bevan et al., Nucl.Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vectorcan then be used in known manner for the transformation of plants, suchas plants used as a model, like Arabidopsis or crop plants such as, byway of example, tobacco plants, for example by immersing bruised leavesor chopped leaves in an agrobacterial solution and then culturing themin suitable media. The transformation of plants by means ofAgrobacterium tumefaciens is described, for example, by Hofgen andWillmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter aliafrom F. F. White, Vectors for Gene Transfer in Higher Plants; inTransgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kungand R. Wu, Academic Press, 1993, pp. 15-38.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganization. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a T6PP protein as defined hereinabove. Preferred hostcells according to the invention are plant cells.

Host plants for the nucleic acids or the vector used in the methodaccording to the invention, the expression cassette or construct orvector are, in principle, advantageous in all plants, which are capableof synthesizing the polypeptides used in the inventive method.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic plants are mentionedherein.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, ears, flowers, stems, and other biologicalsamples. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch,proteins, or an extract derived from corn event MZDT09Y. The biologicalsample or extract is selected from the group consisting of corn flour,corn meal, corn syrup, corn oil, corn starch, and cereals manufacturedin whole or in part to contain corn-by-products.

Reference herein to enhanced yield-related traits is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground. In particular, such harvestable parts are seeds, and performanceof the methods of the invention results in plants having increased seedyield relative to the seed yield of suitable control plants.

The terms “increase”, “improving” or “improve” are interchangeable andshall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9%or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or40% more yield and/or growth in comparison to the wild type plant asdefined herein.

Increased seed yield may manifest itself as one or more of thefollowing: a) an increase in seed biomass (total seed weight) which maybe on an individual seed basis and/or per plant and/or per hectare oracre; b) increased number of flowers per plant; c) increased number of(filled) seeds; d) increased seed filling rate (which is expressed asthe ratio between the number of filled seeds divided by the total numberof seeds); e) increased harvest index, which is expressed as a ratio ofthe yield of harvestable parts, such as seeds, divided by the totalbiomass; and f) increased thousand kernel weight (TKW), which isextrapolated from the number of filled seeds counted and their totalweight. An increased TKW may result from an increased seed size and/orseed weight, and may also result from an increase in embryo and/orendosperm size.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established perhectare or acre, an increase in the number of ears per plant, anincrease in the number of rows, number of kernels per row, kernelweight, thousand kernel weight, ear length/diameter, increase in theseed filling rate (which is the number of filled seeds divided by thetotal number of seeds and multiplied by 100), among others. Taking riceas an example, a yield increase may manifest itself as an increase inone or more of the following: number of plants per hectare or acre,number of panicles per plant, number of spikelets per panicle, number offlowers (florets) per panicle (which is expressed as a ratio of thenumber of filled seeds over the number of primary panicles), increase inthe seed filling rate (which is the number of filled seeds divided bythe total number of seeds and multiplied by 100), increase in thousandkernel weight, among others.

Since the corn event MZDT09Y plants according to the present inventionhave increased yield, it is likely that these plants exhibit anincreased growth rate (during at least part of their life cycle),relative to the growth rate of control plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts of a plant (including seeds), or may be throughoutsubstantially the whole plant. Plants having an increased growth ratemay have a shorter life cycle. The life cycle of a plant may be taken tomean the time needed to grow from a dry mature seed up to the stagewhere the plant has produced dry mature seeds, similar to the startingmaterial. This life cycle may be influenced by factors such as earlyvigor, growth rate, greenness index, flowering time and speed of seedmaturation. The increase in growth rate may take place at one or morestages in the life cycle of a plant or during substantially the wholeplant life cycle. Increased growth rate during the early stages in thelife cycle of a plant may reflect enhanced vigor. The increase in growthrate may alter the harvest cycle of a plant allowing plants to be sownlater and/or harvested sooner than would otherwise be possible (asimilar effect may be obtained with earlier flowering time). If thegrowth rate is sufficiently increased, it may allow for the furthersowing of seeds of the same plant species (for example sowing andharvesting of rice plants followed by sowing and harvesting of furtherrice plants all within one conventional growing period) Similarly, ifthe growth rate is sufficiently increased, it may allow for the furthersowing of seeds of different plants species (for example the sowing andharvesting of corn plants followed by, for example, the sowing andoptional harvesting of soy bean, potato or any other suitable plant).Harvesting additional times from the same rootstock in the case of somecrop plants may also be possible. Altering the harvest cycle of a plantmay lead to an increase in annual biomass production per acre (due to anincrease in the number of times (say in a year) that any particularplant may be grown and harvested). An increase in growth rate may alsoallow for the cultivation of transgenic plants in a wider geographicalarea than their wild-type counterparts, since the territoriallimitations for growing a crop are often determined by adverseenvironmental conditions either at the time of planting (early season)or at the time of harvesting (late season). Such adverse conditions maybe avoided if the harvest cycle is shortened. The growth rate may bedetermined by deriving various parameters from growth curves, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

Performance of the methods of the invention gives plants having anincreased growth rate relative to control plants. Therefore, accordingto the present invention, there is provided a method for increasing thegrowth rate of plants, which method comprises modulating expression,preferably increasing expression, in a plant of a nucleic acid encodinga T6PP protein as defined herein.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects. Another abiotic stress may result from a nutrientdeficiency, such as a shortage of nitrogen, phosphorus and potassium.

The methods of the present invention may be performed under non-stressconditions or under conditions of mild drought to confer plants havingincreased yield relative to control plants. As reported in Wang et al.(Planta (2003) 218: 1-14), abiotic stress leads to a series ofmorphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinization aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under drought conditions increased yieldrelative to suitable control plants grown under comparable conditions.Therefore, according to the present invention, there is provided amethod for enhancing yield-related traits in plants grown undernon-stress conditions or under drought conditions, which methodcomprises increasing expression in a plant of a nucleic acid encoding amodified T6PP polypeptide having decreased substrate binding and/oractivity.

In one embodiment of the invention, the enhanced yield-related trait ismanifested as an increase in one or more of the following: total numberof seeds per plant, number of filled seeds per plant and seed weight perplant. Preferably, these increases are found in plants grown undernon-stress conditions.

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield- enhancing traits, tolerance to other abiotic andbiotic stresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

EXAMPLES Example 1 Identification and Cloning of the Rice T6PP cDNASequence into a Binary Vector

The first vascular plant trehalose-6-phosphate phosphatase genes werecloned from Arabidopsis thaliana by complementation of a yeast T6PS2deletion mutant (Vogel et al. 1998). The genes designated AtT6PPA andAtT6PPB (GenBank accessions AF007778 and AF007779) were shown at thattime to have trehalose-6-phosphate phosphatase activity. The AtT6PPA andAtTTPB protein sequences were used in TBLASTN queries of maize and ricesequence databases. Sequence alignments organized the hits intoindividual genes. Three maize and three rice T6PP homologs wereidentified. The rice T6PP (OsT6PP) cDNA sequence as indicated by SEQ IDNO: 29 was amplified using high-fidelity PCR. The 50 μL reaction mixtureconsisted of 1 μL rice cDNA library (prepared from callus mRNA inStratagene's Lambda Unizap Vector, primary library size >1×10⁶ pfu,amplified library titer>1×10¹² pfu/mL), 200 μM dNTPs, 1 μL 20 μM ofoligonucleotide primer T6PP-EC-5 (5′-catggaccatggatttgagcaatagctcac-3′)and 1 μL 20 μM of oligonucleotide primer T6PP-EC-3(5′-atcgcagagctcacactgagtgcttcttcc-3′), 5 μL 10×Cloned PFU buffer and2.5 Units of Pfuturbo DNA polymerase. The thermocycling program was 95°C. for 2 minutes followed by 40 cycles of (94° C. for 15 seconds, 50° C.for 1 minute, 72° C. for 1 minute) followed by 72° C. for 10 minutes.The rice T6PP product was cloned with the Zero Blunt TOPO PCR cloningkit. The pCR-Blunt-II-TOPO-OsT6PP is identified by digesting 5 μLpCR-Blunt-II-TOPO-OsT6PP miniprep DNA with EcoRI in a 20 μL reactioncontaining 2 μg BSA and 2 μL 10×EcoRI restriction endonuclease buffer.The reaction is incubated at 37° C. for 2 hours and thepCR-Blunt-II-TOPO-OsT6PP (EcoRI) products are resolved on 1% TAEagarose. The pCR-Blunt-II-TOPO-OsT6PP clone is then sequenced. TheOsT6PP cDNA is flanked by NcoI/SacI restriction endonuclease sites. TheOsT6PP was then further cloned into a binary vector as described inExample 8 of U.S. Patent Application Publication 2007/0006344 (thereinreferred to OsT6PP-3 and indicated by nucleotide SEQ ID NO: 531 andprotein SEQ ID NO: 532)

Example 2 Initial Evaluation of Rice T6PP Maize Events in the Greenhouse

Rice T6PP maize events comprising SEQ ID NO: 1 operably linked to apromoter having preferential expression in maternal reproductive tissue(i.e. OsMADS promoter) were generated and further evaluated in both thegreenhouse and field as described in Examples 8-13 in U.S. PatentApplication Publication 2007/0006344. Initial greenhouse and fieldevaluation of the maize events indicated some events having a yieldincrease in both non-drought and drought conditions (See U.S. PatentApplication Publication 2007/0006344 herein incorporated by reference).

Example 3 Evaluation and Identification High Yielding T6PP Maize Events

The maize events shown to confer a yield increase in the trialsdescribed in Example 2 and more specifically in U.S. Patent ApplicationPublication 2007/0006344, were further characterized for yield and fieldefficacy. These events contained either binary construct 15777 (SEQ IDNO: 30) or 15769 (SEQ ID NO: 31) as is described in U.S. PatentApplication Publication 2007/0006344. Essentially binary construct 15769comprises an expression cassette having a OsT6PP (indicated in SEQ IDNO: 29 of the current application) operably linked to a OsMADS6promoter. Binary construct 15777 contains the same expression cassette(OsMADS6 promoter and OsT6PP coding sequence) with the addition oftranscriptional enhancers upstream of the OsMADS6 promoter. The detailsand specifics of both these constructs may again be found in the U.S.Patent Application Publication 2007/0006344. Overall there were 645 TOmaize events generated from 15769 and 587 maize events were generatedcomprising the 15777 binary construct. Following the course ofgeneration of transgenic events during plant transformation, selectionof events having successfully integrated into the genomic DNA, as wellas growth in greenhouse and field conditions relatively a small numberof events were carried forward for field trials based on the selectioncriteria as well as plant event survival and phenotype criteria. Therelatively high level of attrition led to only 17 events showing fieldefficacy. Events derived from maize plants comprising the 15777 binaryconstruct proved to be most efficacious in the field testing. Twoevents, MZDT09Y and MZDT08H, were selected based upon viability andperformance in managed stress environments (yield preservation underdrought at flowering) and in agronomic trials which measured yield.Overall, best performing events comprising construct 15777 demonstrateda significant bushel per acre yield advantage over control checksamples.

Example 4 Corn Events MZDT09Y and MZDT08H Sequence Analysis

Corn event MZDT09Y and corn event MZDT08H were further analyzed bysequencing of the T6PP CDS. PCR was used to amplify the integratedOsT6PP coding sequence using primers that anneal to the 5′ and 3′ regionof the coding sequence. The respective PCR amplicons resulted in theapproximate 1.1 Kb band size as would be expected from the codingsequence of the OsT6PP as depicted in SEQ ID NO: 29 which was thesequence that was comprised in the relative expression cassette. Theamplicons were further sequenced as is well established in the art.Sequencing data indicated that both events contained modifications.MZDT09Y contained a single point mutation at nucleotide 730 respectiveto SEQ ID NO: 29 (T*CATTA where * indicates point of mutation). Thissingle mutation led to an amino acid mutation changing a His residue toan Asp residue. MZDT08H was found to contain two modifications atnucleotides 305 (TG*CTTCC where * indicates point of mutation) and 388(CGCC*ATT where * indicates point of mutation) respective to SEQ ID NO:29. Interestingly, these mutations are located in highly conserveddomains of the T6PP protein.

Primer pairs used to sequence the heterologous insert are as follows:09Y-IS-1 (SEQ ID NO: 26) was amplified using forward primer 09Y-RBFS-F1(SEQ ID NO: 16) and reverse primer 09Y-IS-R1 (SEQ ID NO: 18); 09Y-IS-2(SEQ ID NO: 27) was amplified using forward primer 09Y-IS-F2 (SEQ ID NO:19) and reverse primer 09Y-IS-R2 (SEQ ID NO: 20); 09Y-IS-3 (SEQ ID NO:28) was amplified using forward primer 09Y-IS-F6 (SEQ ID NO: 21) andreverse primer 09Y-IS-R6 (SEQ ID NO: 22).

The modified T6PP enzyme (SEQ ID NO: 11) of corn event MZDT09Y hasapproximately 10% activity relative to the unmodified T6PP enzyme, andyet corn event MZDT09Y is not affected by yield drag. However, themodified T6PP enzyme of MZDT08H does impart a slight yield drag on thecorn plant. For this surprising and unexpected result, corn eventMZDT09Y was selected for progression.

Example 5 Methods of Detecting Corn Event MZDT09Y by PCR

Corn event MZDT09Y can be detected by assaying for a nucleotide sequencecomprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.Polymerase chain reaction (PCR) is a standard method of amplifying DNApracticed by those of skill in the art, provided that the sequence ofthe template DNA is known. SEQ ID NOs: 1-4 are completely unique toMZDT09Y and are disclosed herein for the first time. Primer pairsdifferent from those primers explicitly disclosed herein may bedeveloped by one of ordinary skill in the art and still achieve the samefunction, that is, to identify corn event MZDT09Y.

Primers SEQ ID NO: 12 (forward primer MZDT09Y-RB1) and SEQ ID NO: 13(reverse primer ESPCR0001) function together in a PCR reaction toproduce a right border amplicon comprising SEQ ID NO: 23 and itscomplementary sequence, which is indicative of the presence of MZDT09Ytemplate DNA. This amplicon also comprises SEQ

ID NO: 1 and SEQ ID NO: 3.

Primers SEQ ID NO: 14 (forward primer 09Y-LBFS-F1) and SEQ ID NO: 15(reverse primer 09Y-LBFS-R4) function together in a PCR reaction toproduce a left border amplicon comprising SEQ ID NO: 24 and itscomplementary sequence, which is indicative of the presence of MZDT09Ytemplate DNA. This amplicon also comprises SEQ ID NO: 2 and SEQ ID NO:4.

In addition to conventional, gel-based PCR, TaqMan® PCR (Invitrogen™)which uses fluorescence to enable detection of amplification inreal-time, may also be used to detect the presence of corn eventMZDT09Y. Forward primer P23198 (SEQ ID NO: 32), reverse primer P23352(SEQ ID NO: 33), and probe P23200 (SEQ ID NO: 34, labeled with FAM onits 5′-terminus, and with BHQ1 on its 3′-terminus) function together ina TaqMan PCR reaction in the presence of MZT09Y template DNA to producean amplicon (SEQ ID NO: 35), and thereby fluorescence, diagnostic ofcorn event MZDT09Y.

Example 6 Methods of Detecting Corn Event MZDT09Y by Hybridization

Corn event MZDT09Y can be detected by assaying using hybridizationtechniques for a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO: 4. Methods of hybridization, for examplea Southern blot, can detect DNA, provided that the sequence of thetemplate DNA is known. SEQ ID NOs: 1-4 are completely unique to MZDT09Yand are disclosed herein for the first time.

By way of example and not limitation, DNA samples are cut withrestriction enzymes and run overnight on an agarose gel in 1×TBE bufferat about 32 volts. Gels are photographed, washed, and blotted onto nylonmembrane with 10×SSC as the transfer solution. They are linked to themembrane with UV light and pre-hybridized with calf thymus DNA at 65° C.Probes comprising SEQ ID NO: 1 or SEQ ID NO: 2 are labeled withradioactive Phosphorus 32. Probes are added and hybridized at 65° C., 3hrs to overnight. Blots are washed several times and exposed in aphosphorimager cassette. Images are developed and scored.

Deposit

Applicants have made a deposit of corn seed of event MZDT09Y disclosedabove on Jun. 28, 2012, in accordance with the Budapest Treaty at theAmerican Type

Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va.,20110, USA under ATCC Accession No. PTA-13025. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or the effective life of the patent, whichever islonger, and will be replaced as necessary during that period. Applicantsimpose no restrictions on the availability of the deposited materialfrom the ATCC; however, Applicants have no authority to waive anyrestrictions imposed by law on the transfer of biological material orits transportation in commerce. Applicants do not waive any infringementof their rights granted under this patent or under the Plant VarietyProtection Act (7 USC 2321 et seq.).

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent document was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be clear to those of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

1. A nucleic acid molecule, preferably isolated, comprising a nucleotidesequence that is unique to event MZDT09Y, wherein the nucleotidesequence is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof.
 2. Theisolated nucleic acid molecule according to claim 1, wherein the nucleicacid molecule is comprised in a corn seed deposited at the American TypeCulture Collection under the accession number PTA-13025.
 3. A pair ofpolynucleotide primers comprising a first polynucleotide primer and asecond polynucleotide primer which function together in the presence ofan event MZDT09Y DNA template in a sample to produce an amplicondiagnostic for event MZDT09Y.
 4. The pair of polynucleotide primersaccording to claim 3, wherein a. a sequence of the first polynucleotideprimer or a sequence of the second polynucleotide primer is chosen fromSEQ ID NO: 7, or the complement thereof; or b. a sequence of the firstpolynucleotide primer is or is complementary to a corn plant genomesequence flanking the point of insertion of a heterologous DNA sequenceinserted into the corn plant genome of event MZDT09Y, and a sequence ofthe second polynucleotide primer is or is complementary to theheterologous DNA sequence inserted into the genome of event MZDT09Y. 5.The pair of polynucleotide primers according to claim 4, wherein a. thefirst polynucleotide primer comprises at least 10 contiguous nucleotidesof a nucleotide sequence selected from the group consisting of SEQ IDNO: 5, SEQ ID NO: 6, and the complements thereof; and b. the secondpolynucleotide primer comprises at least 10 contiguous nucleotides fromSEQ ID NO: 7, or the complements thereof.
 6. The pair of polynucleotideprimer according for claim 5, wherein a. the first polynucleotide primercomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 32, and the complementsthereof; and b. the second polynucleotide primer comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 33, and the complements thereof.
 7. The pair ofpolynucleotide primers according to claim 6, wherein a. the firstpolynucleotide primer consists of SEQ ID NO: 12 and the secondpolynucleotide primer consists of SEQ ID NO: 13; or b. the firstpolynucleotide primer consists of SEQ ID NO: 14 and the secondpolynucleotide primer consists of SEQ ID NO: 15; or c. the firstpolynucleotide primer consists of SEQ ID NO: 32 and the secondpolynucleotide primer consists of SEQ ID NO:
 33. 8. A method ofdetecting the presence of a nucleic acid molecule that is unique toevent MZDT09Y in a sample comprising corn nucleic acids, the methodcomprising: a. contacting the sample with a pair of primers that, whenused in a nucleic acid amplification reaction with genomic DNA fromevent MZDT09Y produces an amplicon that is diagnostic for event MZDT09Y;b. performing a nucleic acid amplification reaction, thereby producingthe amplicon; and c. detecting the amplicon.
 9. A method of detectingthe presence of a nucleic acid molecule that is unique to event MZDT09Yin a sample comprising corn nucleic acids, the method comprising: a.contacting the sample with a probe that hybridizes under high stringencyconditions with genomic DNA from event MZDT09Y and does not hybridizeunder high stringency conditions with DNA of a control corn plant; b.subjecting the sample and probe to high stringency hybridizationconditions; and c. detecting hybridization of the probe to the nucleicacid molecule.
 10. A kit for detecting nucleic acids that are unique toevent MZDT09Y comprising at least one nucleic acid molecule ofsufficient length of contiguous polynucleotides to function as a primeror probe in a nucleic acid detection method, and which uponamplification of or hybridization to a target nucleic acid sequence in asample followed by detection of the amplicon or hybridization to thetarget sequence, are diagnostic for the presence of nucleic acidsequences unique to event MZDT09Y in the sample.
 11. The kit accordingto claim 10, wherein the nucleic acid molecule of sufficient length ofcontinuous polynucleotides comprises a nucleotide sequence selected fromthe group consisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 8, and the complements thereof.
 12. The kit accordingto claim 11, wherein the nucleic acid molecule is selected from thegroup consisting of SEQ ID NOs: 12-15, SEQ ID NOs: 32-34, and thecomplements thereof.
 13. A transgenic corn plant, or cells or tissuesthereof, comprising a nucleic acid molecule according to claim
 1. 14. Acorn seed comprising a nucleic acid molecule according to claim 1, anexample of the seed being deposited at the American Type CultureCollection under the accession number PTA-13025.
 15. A biological samplederived from an event MZDT09Y corn plant, tissue, or seed, wherein thesample comprises a nucleotide sequence which is or is complementary to anucleotide sequence of claim 1 and wherein the sequence is detectable inthe sample using a nucleic acid amplification or nucleic acidhybridization method.
 16. The biological sample of claim 15 wherein thesample is selected from the group consisting of corn flour, corn meal,corn syrup, corn oil, corn starch, and cereals manufactured in whole orin part to contain corn by-products.
 17. An extract derived from thebiological sample according to claim
 15. 18. The extract of claim 17wherein the sample is selected from the group consisting of corn flour,corn meal, corn syrup, corn oil, corn starch, and cereals manufacturedin whole or in part to contain corn by-products.
 19. A method forproducing a corn plant with increased yield comprising: a. sexuallycrossing a first parent corn plant with a second parent corn plant,wherein said first or second parent corn plant comprises event MZDT09YDNA, thereby producing a plurality of first generation progeny plants;b. selecting a first generation progeny plant with increased yield; c.selfing the first generation progeny plant, thereby producing aplurality of second generation progeny plants; and d. selecting from thesecond generation progeny plants, a plant with increased yield; whereinthe second generation progeny plants comprise the nucleic acid moleculeaccording to claim
 1. 20. The method of claim 19, wherein increasedyield is indicated by an increase as compared to a control plant of anyone of the following: increased grain yield, increased seed, increasedseed weight, increased biomass, increased sugar, increased oil,increased plant vigor, increased yield under non-optimal conditions,increased yield under stress conditions and increased yield under waterstress conditions.
 21. A method for producing a corn plant withincreased tolerance to abiotic stress comprising: a. sexually crossing afirst parent corn plant with a second parent corn plant, wherein saidfirst or second parent corn plant comprises event MZDT09Y DNA, therebyproducing a plurality of first generation progeny plants; b. selecting afirst generation progeny plant with increased tolerance to abioticstress; c. selfing the first generation progeny plant, thereby producinga plurality of second generation progeny plants; and d. selecting fromthe second generation progeny plants, a plant with increase tolerance toabiotic stress; wherein the second generation progeny plants comprisethe nucleic acid molecule according to claim
 1. 22. The method of claim21, wherein said abiotic stress comprises stress selected from the groupconsisting of water stress, heat stress or cold stress.
 23. The methodof claim 22, wherein water stress is caused by drought.
 24. A method ofproducing hybrid corn seeds comprising: a. planting seeds of a firstinbred corn line comprising event MZDT09Y and seeds of a second inbredline having a genotype different from the first inbred corn line; b.cultivating corn plants resulting from said planting until time offlowering; c. emasculating said flowers of plants of one of the corninbred lines; d. sexually crossing the two different inbred lines witheach other; and e. harvesting the hybrid seed produced thereby; whereinthe hybrid seed comprise the nucleic acid molecule according to claim 1.25. The method according to claim 24, wherein the plants of the firstinbred corn line are the female parents or male parents.
 26. Hybrid seedproduced by the method of claim 24.